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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is comparable to OECD Guideline 471 with acceptable restrictions (no 2nd trial for verification of weak positive effects at dose levels between 2 and 5 mg/plate).

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Mutagenicity Test Data of Existing Chemical Substances
Author:
JETOC
Year:
1996
Bibliographic source:
Edited and Published January 1996 by Japan Chemical Industry Ecology-Toxicology & Information Center, Japan
Reference Type:
publication
Title:
Mutagenicity Test Data of Existing Chemical Substances
Author:
JETOC
Year:
1997
Bibliographic source:
Edited and Published February 1997 by Japan Chemical Industry Ecology-Toxicology & Information Center, Japan

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
JETOC 1996
Purity: 99.2%, source: Wako Pure Chem. Japan, no further data
JETOC 1997
Purity 98.0%, source: Aldrich Chemicals Inc.; Lot no.: 16520P X; no further data

Method

Target gene:
His- in Salmonella typhimurium
Trp- in E. coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
plus E. coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbital i.p. injected into male Sprague-Dawley rats (200 g) at a dosage of 30 mg/kg bw/day for 4 days before sacrifice; liver S9 prepared and mixed with supplements.
Test concentrations with justification for top dose:
JETOC 1996: 0, 50, 100, 200, 500, 1000, 2000, 5000 µg/plate.
JETOC 1997: 0, 0.08, 0.3, 1.2, 4.9, 19.5, 78, 313, 1250, 5000 µg/plate.
5 mg/plate is the max. recommended concentration according to OECD 471.
Vehicle / solvent:
DMSO in both experiments
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
JETOC tested 2-chloroethanol 1996 and 1997 under similar experimental conditions but different sources for the test substance. Both results are presented in this data sheet.
The authors used the preincubation method (Matsushima et al., 1980). All plates were incubated for 48 hours at 37°C and the numbers of revertant colonies were scored.

Determination of cytotoxicity
Growth of the background lawn of tester strains on each plate examined under a stereo microscope. The decrease in back-ground lawn is a measure for bactericidal effects (decrease in the number of micro colonies and an increase in their diameter).

Positive control
AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, used without S9-mix in TA98 (0.1 µg/plate), WP2uvrA (0.01 µg/plate) and in TA100 (0.01 µg/plate)
NaN3: sodium azide, used withot S9-mix in TA1535 (0.5 µg/plate)
9AA : 9-aminoacridine, used without S9-mix in TA1537 (80 µg/plate)
2AA : 2-amino anthracene, used with S9-mix in all strains at concentrations between 0.5 µg/plate (TA98) and 10 µg/plate (WP2uvrA).
Evaluation criteria:
Positive:
1) dose-dependent effects
2) at least 2-fold increase in revertants
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
(tested up to 5 mg/plate)
Vehicle controls validity:
other: some values not within the historical vehicle controls (see Table below)
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
but only at 5 mg/plate; negative results with S9-mix
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
other: slightly higher than historical vehicle control (see Table below)
Positive controls validity:
valid
Species / strain:
other: TA1537, TA98, TA100, WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 5 mg/plate without S9-mix; tested up to limit concentrations)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
but only at 5 mg/plate; negative results without S9-mix
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A summary Table of results is presented below.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Precipitation: no data, but no precipitation expected

COMPARISON WITH HISTORICAL CONTROL DATA:
on page 33 (Table 2) historical control data were given; these data were also presented in the Table below


ADDITIONAL INFORMATION ON CYTOTOXICITY: see Table below
Remarks on result:
other: other: 1st test in JETOC1996
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Ames test in JETOC 1996

Revertants per plate, mean of of two plates given, cytotoxicity marked by *

Dose in µg per plate

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Historical vehicle

138+-27

139+-29

14+-5

13+-4

29+-11

34+-11

20+-8

26+-7

8+-2

11+-4

Current vehicle

142

152

22

17

43

47

17

25

4

7

50

156

162

21

16

44

55

13

32

6

6

100

148

164

17

14

33

38

16

25

8

5

200

151

166

11

14

45

52

21

30

6

8

500

148

172

18

11

40

50

17

42

4

4

1000

138

168

20

15

39

58

11

33

6

4

2000

158

143

20

15

65

64

21

38

6

5

5000

198

171

29

25

186

79

24

33

7

7

Positive control

635

1455

315

510

553

1022

448

479

600

354

x

x

Ames test in JETOC 1997

Revertants per plate, mean of of two plates given, cytotoxicity marked by *

Dose in µg per plate

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Historical vehicle

138+-27

139+-29

14+-5

13+-4

29+-11

34+-11

20+-8

26+-7

8+-2

11+-4

Current vehicle

149

163

15

16

32

39

19

27

11

13

0.08

168

166

19

17

33

44

21

25

7

13

0.3

161

157

19

14

34

38

21

25

12

17

1.2

149

148

15

19

32

36

20

18

10

14

4.9

175

169

12

15

28

35

12

28

9

12

19.5

164

175

17

15

28

39

13

26

6

15

78

160

171

16

16

26

44

19

25

9

16

313

183

168

12

13

31

39

22

23

14

14

1250

165

185

14

24

38

43

19

16

9

14

5000

203*

167

20*

37

52

65

25*

20

11*

10

Positive control

765

920

362

242

177

917

510

203

471

177

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: weak positive

Weak positive results only at a dose level of 5 mg/plate (limit concentration) which are not reproducible in independent experiments.
Executive summary:

The study is comparable to OECD Guideline 471 with accepatable restrictions (no 2nd trial for verification of weak positive effects at dose levels between 2 and 5 mg/plate).

In the Ames test on S. typhimurium TA1535, TA1537, TA98, TA100, and E. coli WP2uvrA the bacteria were exposed with and without metabolic activation in a first test (JETOC 1996) to 0, 50, 100, 200, 500, 1000, 2000, 5000 µg/plate and in a 2nd test (JETOC 1997) to 0, 0.08, 0.3, 1.2, 4.9, 19.5, 78, 313, 1250, 5000 µg/plate. In the first test an increase in the number of revertants was detected only in WP2uvrA without S9 -mix at the highest dose level of 5 mg/plate which is the limit concentration according to OECD 471. Negative results in WP2uvrA were obtained in a 2nd test (JETOC 1997) at the limit dose level of 5 mg/plate. In the 2nd test weak positive results were found only in TA1535 with S9 -mix, again at the high dose level of 5 mg/plate. Negative results were seen in the 1st test in this strain at the same concentration.

Conclusion: Weak positive results only at a dose level of 5 mg/plate (limit concentration) which are not reproducible in independent tests.